Population-based proteomics: Platforms to advance diagnosis and treatment of metabolic liver diseases
Authors: Kara Wegermann, Anna Mae Diehl
Journal: Hepatology (2023)

A critical function of the adult liver is the maintenance of systemic energy homeostasis. As such, the liver is a major regulator of the plasma proteome. Qualitative and/or quantitative changes in the plasma proteome provide insight into the causes and consequences of metabolic stress, including hepatic steatosis and fibrosis. In this issue of Hepatology, Abozaid et al1 sought to define how the proteome changes with hepatic steatosis and fibrosis to elucidate fatty liver disease pathogenesis and identify potential biomarkers of fatty liver disease severity. Multiplex platforms were used to quantify large panels of proteins associated with either inflammation or cardiometabolic stress in blood samples from over 2500 participants in the population-based Rotterdam study who fulfilled enrollment criteria for fatty liver disease due to NAFLD or metabolic-associated liver disease (MAFLD). Results were correlated with the presence/absence of liver steatosis and fibrosis severity after considering potentially contributing factors (eg, body mass index, type 2 diabetes, alcohol consumption, and cigarette smoking). Liver diagnoses were established retrospectively by ultrasound and elastography on an average of 5 years after blood was obtained for the proteomic analyses. About 10% (8 of 83) of the inflammation-associated proteins correlated with the presence of steatosis on ultrasound exam, and ~22% (19 of 88) of the cardiometabolic risk-associated proteins correlated with fibrosis severity as quantified by elastography. More than half (15/27, 56%) of the liver disease-associated proteins correlated with both steatosis and fibrosis diagnoses, and most of these proteins (80%) positively correlated with liver abnormalities. The 2 proteins that exhibited the strongest correlations with both fat and fibrosis were FGF21 and carboxylesterase 1. Signaling pathway analysis linked proteome changes with statistically significant changes in noncanonical NF-kB signaling and coagulation. Two of the 15 conserved disease-associated proteins, IL18-R1 and carboxylesterase 1, changed significantly in hepatocyte spheroids that were treated with fatty acids to model lipotoxic stress, suggesting that they were generated by fatty hepatocytes in NAFLD/MAFLD subjects. Receiver operator curve analyses demonstrated that the plasma proteome data was superior to fibrosis-4 for the diagnosis of advanced (F2 or greater) fibrosis but no better than a sonogram or fat index for diagnosing steatosis. These studies forecast approaches that will be fine-tuned and applied to discover new biomarkers and therapeutic targets in metabolic liver diseases. Strengths of the present work include its large population-based study cohort, the application of assays that globally profile plasma proteins that associate with inflammatory and cardiometabolic stress (mechanisms implicated in NAFLD/MAFLD pathogenesis), and efforts to compare the diagnostic performance of the proteome-based protein panel, with current blood-based algorithms for diagnosing fatty liver disease (fat index) and staging fibrosis severity (fibrosis-4). The authors appropriately acknowledge and justify the main limitations of their study, that is, the primary tests for liver disease diagnosis were done several years after the blood sample that was used for proteomic analysis and not confirmed by histology; the proteome assays were restricted to proteins that could be detected by the 2 multiplex platforms; and liver spheroids are not ideal tools for validating pathobiological implications as they do not fully recapitulate liver tissues or physiologically relevant metabolic stress. Mindful of these caveats, readers will understand that further research is needed to determine the ultimate significance of the specific protein biomarkers identified by the present study. It will be particularly important to cross-reference these proteome findings with proteome results derived from other studies that used different platforms and/or evaluated blood samples from different patient cohorts. Presumably, if changes in the plasma proteome reflect underlying pathobiology in the liver, conserved pathways will be identified across different platforms and cohorts. However, a cursory comparison of the findings in the present study with those reported by 2 recent publications that used different assays to assess the plasma proteomes of small cohorts of patients with biopsy-proven NAFLD2,3 did not identify overlap in specific disease-related proteins across studies; further investigation is needed to determine whether signaling pathway dysregulation was conserved. Combining plasma proteomic data with genetic and transcriptomic analyses may provide novel strategies for diagnosis and/or risk stratification. For example, a recent study of patients from Iceland and in the UK Biobank integrated proteome findings with genome-wide association studies and liver transcriptomic data. The authors found that 273 circulating proteins were associated with genetic variants linked to NAFL. Eight proteins were each associated with 5 or more genetic variants and also with NAFL or cirrhosis. A signature incorporating genetic risk scores and proteomic data along with clinical data was able to discriminate between NAFL and cirrhosis with high accuracy. Taken together, these data suggest that circulating proteins could serve as novel biomarkers for liver disease, augmenting or even replacing genetic data in some situations.4 It remains conceivable, however, that other factors, such as lipids or smaller metabolites, may be superior to proteins as biomarkers of metabolic liver disease. Finally, the fact that levels of certain plasma proteins correlated with NAFLD/MAFLD severity in the present study does not establish a causal role for the protein(s) in liver disease pathogenesis. For example, FGF21 was the most strongly correlated protein with liver disease severity in the present study, and it is thought to play a hepatoprotective role in NAFLD/MAFLD.5 Nonetheless, it is intriguing that changes in plasma levels of several clotting cascade-related proteins correlated with the diagnosis of fibrosing NAFLD/MAFLD, given the evidence that hepatic microvascular thrombosis has long been implicated in fatty liver disease pathogenesis6 and dysregulated clotting associates, with metabolic syndrome-related pathology in other organs.7–9 In summary, hepatology is entering a new era of discovery thanks to the increasing availability of assays that enable global and unbiased profiling of bio-samples from large, well-characterized human populations at risk for liver disease. The resulting knowledge about disease pathogenesis, coupled with new diagnostic and prognostic tests, will help to individualize and optimize the management of patients with metabolic liver disease.